Proteomic Atlas of Atherosclerosis: The Contribution of Proteoglycans to Sex Differences, Plaque Phenotypes, and Outcomes.

BACKGROUND: Using proteomics, we aimed to reveal molecular types of human atherosclerotic lesions and study their associations with histology, imaging, and cardiovascular outcomes. METHODS: Two hundred nineteen carotid endarterectomy samples were procured from 120 patients. A sequential protein extraction protocol was employed in conjunction with multiplexed, discovery proteomics. To focus on extracellular proteins, parallel reaction monitoring was employed for targeted proteomics. Proteomic signatures were integrated with bulk, single-cell, and spatial RNA-sequencing data, and validated in 200 patients from the Athero-Express Biobank study. RESULTS: This extensive proteomics analysis identified plaque inflammation and calcification signatures, which were inversely correlated and validated using targeted proteomics. The inflammation signature was characterized by the presence of neutrophil-derived proteins, such as S100A8/9 (calprotectin) and myeloperoxidase, whereas the calcification signature included fetuin-A, osteopontin, and gamma-carboxylated proteins. The proteomics data also revealed sex differences in atherosclerosis, with large-aggregating proteoglycans versican and aggrecan being more abundant in females and exhibiting an inverse correlation with estradiol levels. The integration of RNA-sequencing data attributed the inflammation signature predominantly to neutrophils and macrophages, and the calcification and sex signatures to smooth muscle cells, except for certain plasma proteins that were not expressed but retained in plaques, such as fetuin-A. Dimensionality reduction and machine learning techniques were applied to identify 4 distinct plaque phenotypes based on proteomics data. A protein signature of 4 key proteins (calponin, protein C, serpin H1, and versican) predicted future cardiovascular mortality with an area under the curve of 75% and 67.5% in the discovery and validation cohort, respectively, surpassing the prognostic performance of imaging and histology. CONCLUSIONS: Plaque proteomics redefined clinically relevant patient groups with distinct outcomes, identifying subgroups of male and female patients with elevated risk of future cardiovascular events.

Aberrant DNA Methylation, Expression, and Occurrence of Transcript Variants of the ABC Transporter ABCA7 in Breast Cancer.

The ABC transporter ABCA7 has been found to be aberrantly expressed in a variety of cancer types, including breast cancer. We searched for specific epigenetic and genetic alterations and alternative splicing variants of ABCA7 in breast cancer and investigated whether these alterations are associated with ABCA7 expression. By analyzing tumor tissues from breast cancer patients, we found CpGs at the exon 5-intron 5 boundary aberrantly methylated in a molecular subtype-specific manner. The detection of altered DNA methylation in tumor-adjacent tissues suggests epigenetic field cancerization. In breast cancer cell lines, DNA methylation levels of CpGs in promoter-exon 1, intron 1, and at the exon 5-intron 5 boundary were not correlated with ABCA7 mRNA levels. By qPCR involving intron-specific and intron-flanking primers, we identified intron-containing ABCA7 mRNA transcripts. The occurrence of intron-containing transcripts was neither molecular subtype-specific nor directly correlated with DNA methylation at the respective exon-intron boundaries. Treatment of breast cancer cell lines MCF-7, BT-474, SK-BR3, and MDA-MB-231 with doxorubicin or paclitaxel for 72 h resulted in altered ABCA7 intron levels. Shotgun proteomics revealed that an increase in intron-containing transcripts was associated with significant dysregulation of splicing factors linked to alternative splicing.